SPECTROSCOPIC PROPERTIES OF SUBTILISIN DY SELECTIVELY MODIFIED WITH FLUORESCEIN ISOTHIOCYANATE

In this work we describe spectroscopic properties of subtilisin DY sel ectively labeled at alanine I by fluorescein isothiocyanate. The react ion takes place in dark at pH 9.3, 20 degrees C, within a 4 h period, in the presence of 5-fold molar excess of the reagent over the protein . As a result, 1.0 mol of fluorescein mol(-1) was covalently bound to the alpha-amino group of subtilisin DY. Circular dichroism studies sho wed that the modification created some conformational changes of the p roteinase polypeptide chain and the proteolytic activity decreased up to nearly 70% of the control. FTC-subtilisin DY retained a highly orde red structure with 82% of the alpha-helix structure of the native enzy me. Singlet-singlet energy transfer between the tyrosyl residues of su btilisin DY (donors) and the covalently bound dye (acceptor) was obser ved. Fluorescence measurements demonstrated that 30% of the light abso rbed by the phenolic groups is transfered to the fluorescein group aft er excitation at 280 nm. An average distance of 28.7 +/- 2 Angstrom be tween the emitting tyrosyl side chains and the bound fluorescein was c alculated from energy transfer data. The labeled proteinase can be det ected at concentrations as low as 10(-7) M. (C) 1997 Elsevier Science B.V.